Sphingosine kinase 1 promotes liver fibrosis by preventing miR-19b-3p-mediated inhibition of CCR2.

Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) leads to liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the sphingosine kinase (SphK)1-dependent effect on liver fibrosis. The levels of expression and activity of SphK1 were significantly increased in fibrotic livers compared with the normal livers in human. SphK1 was coexpressed with a range of HSC/KC markers including desmin, α-smooth muscle actin (α-SMA) and F4/80 in fibrotic liver. Deficiency of SphK1 (SphK1-/- ) resulted in a marked amelioration of hepatic injury, including transaminase activities, histology, collagen deposition, α-SMA and inflammation, in CCl4 or bile duct ligation (BDL)-induced mice. Likewise, treatment with a specific inhibitor of SphK1, 5C, also significantly prevented liver injury and fibrosis in mice induced by CCl4 or BDL. In cellular levels, inhibition of SphK1 significantly blocked the activation and migration of HSCs and KCs. Moreover, SphK1 knockout in KCs reduced the secretion of CCL2, and SphK1 knockout in HSCs reduced C-C motif chemokine receptor 2 ([CCR2] CCL2 receptor) expression in HSCs. CCL2 in SphK1-/- mice was lower whereas microRNA-19b-3p in SphK1-/- mice was higher compared with wild-type (WT) mice. Furthermore, microRNA-19b-3p downregulated CCR2 in HSCs. The functional effect of SphK1 in HSCs on liver fibrosis was further strengthened by the results of animal experiments using a bone marrow transplantation (BMT) method. SphK1 has distinct roles in the activation of KCs and HSCs in liver fibrosis. Mechanistically, SphK1 in KCs mediates CCL2 secretion, and SphK1 in HSCs upregulates CCR2 by downregulation of miR-19b-3p. (Hepatology 2018).

Lan T ，Li C ，Yang G ，Sun Y ，Zhuang L ，Ou Y ，Li H ，Wang G ，Kisseleva T ，Brenner D ，Guo J ... -  《-》

Shugan Jianpi Formula attenuate liver fibrosis via regulation of miR-193a-3p/TGF-β2 in hepatic stellate cells: An in vivo and in vitro study.

The Chinese herbal medicine Shugan Jianpi Formula (SGJPF) has traditionally been used to treat various chronic liver disorders. Previous studies have indicated that SGJPF inhibits hepatic stellate cells (HSCs) activation in rats with liver fibrosis (LF) and that miR-193a-3p may be a crucial molecule in LF. However, the mechanisms by which SGJPF regulates HSCs activation through miR-193a-3p remain unclear. This study aimed to determine whether the effect of SGJPF on LF is related to its regulation of miR-193a-3p and TGF-β2, both in a carbon tetrachloride (CCl4)-induced LF mouse model and in TGF-β1-induced JS-1 cells. A CCl4-induced LF mouse model was established to evaluate the anti-fibrotic efficacy of SGJPF by examining liver histopathological changes, collagen deposition, and the expression of α-smooth muscle actin (α-SMA) and collagen-I. To investigate the role of miR-193a-3p in HSCs activation, miR-193a-3p mimics and inhibitors were transfected into TGF-β1-induced JS-1 cells. The potential targets of miR-193a-3p were identified using miRDB, TargetScan 8.0, RNA-seq, and dual-luciferase reporter assays. Finally, the effects of SGJPF on HSCs activation and the miR-193a-3p/TGF-β2 axis were assessed in TGF-β1-treated JS-1 cells using CCK-8, EDU, scratch, RT-qPCR, and Western blotting assays. SGJPF significantly reduced liver damage and fibrosis, inhibited HSCs activation, decreased TGF-β2 levels, and increased miR-193a-3p expression in CCl4-induced LF tissue. Additionally, miR-193a-3p was upregulated in HSCs transfected with miR-193a-3p mimics and downregulated in those with miR-193a-3p inhibitors. High levels of miR-193a-3p, combined with miRNA mimics, inhibited HSCs activation, proliferation, and migration. TGF-β2, a target negatively regulated by miR-193a-3p, partially reversed the effects of miR-193a-3p on TGF-β1-induced HSCs activation. SGJPF also reduced HSCs activation, proliferation, and migration in TGF-β1-treated JS-1 cells. Moreover, treatment with SGJPF-containing serum and miR-193a-3p inhibition restored HSCs activation, proliferation, and migration in TGF-β1-induced JS-1 cells. This study demonstrates that SGJPF ameliorates CCl4-induced liver fibrosis, which is associated with the regulation of miR-193a-3p and TGF-β2 in HSCs. These findings provide a new pharmacological basis for SGJPF and suggest a novel strategy for treating LF through TCM by regulating miRNAs.

Zhou Q ，Zhang X ，Chen S ，Fan C ，Wan K ，Wu C ，Wang X ，Zhang W ，Jiang H ... -  《-》

miR-324-3p Suppresses Hepatic Stellate Cell Activation and Hepatic Fibrosis Via Regulating SMAD4 Signaling Pathway.

In hepatic fibrosis (HF), hepatic stellate cells (HSCs) form the extracellular matrix (ECM), and the pathological accumulation of ECM in the liver leads to inflammation. Our previous research found that miR-324-3p was down-regulated in culture-activated human HSCs. However, the precise effect of miR-324-3p on HF has not been elucidated. In this study, the HF mouse models were induced through directly injecting carbon tetrachloride (CCl4) into mice; the HF cell models were constructed using TGF-β1-treated LX-2 cells. Next, real-time-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and immunohistochemistry (IHC) were applied to assess the expression levels of miR-324-3p, α-smooth muscle actin (α-SMA), Vimentin or SMAD4; hematoxylin and eosin (H&E), Masson' s trichrome and Sirius red staining to evaluate the liver injury; luciferase reporter assay to verify the targeting relationship between miR-324-3p and SMAD4; enzyme-linked immunosorbent assay (ELISA) to determine the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and cell counting kit-8 (CCK-8) and flow cytometry to evaluate the effects of miR-324-3p on cell proliferation and cycle/apoptosis, respectively. The experimental results showed a reduction in miR-324-3p level in CCl4-induced HF mice as well as transforming growth factor (TGF)-β1-activated HSCs. Interestingly, the miR-324-3p level was rescued following the HF recovery process. In HF mice induced by CCl4, miR-324-3p overexpression inhibited liver tissue damage, decreased serum ALT and AST levels, and inhibited fibrosis-related biomarkers (α-SMA, Vimentin) expression, thereby inhibiting HF. Similarly, miR-324-3p overexpression up-regulated α-SMA and Vimentin levels in HF cells, while knockdown of miR-324-3p had the opposite effect. Besides, miR-324-3p played an antifibrotic role through inhibiting the proliferation of hepatocytes. Further experiments confirmed that miR-324-3p targeted and down-regulated SMAD4 expression. SMAD4 was highly expressed in HF cells, and silencing SMAD4 significantly decreased the α-SMA and Vimentin levels in HF cells. Collectively, the miR-324-3p may suppress the activation of HSCs and HF by targeting SMAD4. Therefore, miR-324-3p is identified as a potential and novel therapeutic target for HF.

Chen SY ，Chen X ，Zhu S ，Xu JJ ，Li XF ，Yin NN ，Xiao YY ，Huang C ，Li J ... -  《-》

Catalpol ameliorates liver fibrosis via inhibiting aerobic glycolysis by EphA2/FAK/Src signaling pathway.

Hepatic fibrosis is a pathological process in a variety of acute or chronic liver injuries. Catalpol (CAT), an iridoid glycoside found in Rehmannia glutinosa, has several pharmacological properties, including anti-inflammatory, antidiabetic and anti-fibrotic effects. Nevertheless, there is currently no report on whether CAT regulates the aerobic glycolysis of hepatic stellate cells (HSCs) to inhibit liver fibrosis. This study aimed to investigate the protective effects of CAT on hepatic fibrosis and elucidate its underlying mechanisms. To explore whether CAT improved liver fibrosis in vivo and in vitro, hepatic fibrosis was induced to mice by intraperitoneally injecting carbon tetrachloride (CCl4). Additionally, LX-2 cells were stimulated with transforming growth factor-β (TGF-β) to simulate fibrosis in vitro. Serum markers of liver injury were examined by using an automatic biochemical analyzer. Histopathological staining, Immunofluorescence (IF) staining, Western blot (WB) analysis, co-immunoprecipitation (Co-IP), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), etc. were employed to identify the targeting between CAT and EphA2 and detect the expression of aerobic glycolysis related proteins, fiber markers and signaling pathways that are responsible for CAT's anti-fibrotic effects of CAT. Results showed that CAT significantly inhibited hepatic injury, fibrogenesis and inflammation in mice treated with CCl4. This was demonstrated by the enhancement of fibrosis markers, liver function indices, and histopathology. In addition, CAT significantly inhibited the activation of HSCs in TGF-β-induced LX-2 cells, as indicated by decreased proliferation, migration, and expression of collagen I and a-SMA. The study results also suggested that CAT may exert anti-fibrotic effects by inhibiting glycolysis in activated HSCs and in CCl4-treated mice. Mechanistically, CAT directly targets Ephrin type-A receptor 2 (EphA2) to reduce binding with focal adhesion kinases (FAK) and significantly inhibits the FAK/Src pathway. In addition, the pharmacological inhibition of EphA2 cannot further increase the therapeutic effects of CAT on liver fibrosis in vivo and in vitro. The study findings generally demonstrated that CAT presented a novel therapeutic method to treat hepatic fibrosis; this method which inhibits the aerobic glycolysis of activated HSCs through the EphA2/FAK/Src signaling pathway.

Zhang Q ，Ran T ，Li S ，Han L ，Chen S ，Lin G ，Wu H ，Wu H ，Feng S ，Chen J ，Zhang Q ，Zhao X ... -  《-》

Human induced pluripotent stem cell-derived extracellular vesicles reduce hepatic stellate cell activation and liver fibrosis.

Povero D ，Pinatel EM ，Leszczynska A ，Goyal NP ，Nishio T ，Kim J ，Kneiber D ，de Araujo Horcel L ，Eguchi A ，Ordonez PM ，Kisseleva T ，Feldstein AE ... -  《JCI Insight》